Unveiling the therapeutic potential of Dl-3-n-butylphthalide in NTG-induced migraine mouse: activating the Nrf2 pathway to alleviate oxidative stress and neuroinflammation

Chronic migraine represents a debilitating neurological condition affecting approximately 2% of the general population. Individuals afflicted by chronic migraines endure headaches at least 15 days per month, accompanied by visual, auditory, and olfactory disturbances, as well as symptoms like nausea and vomiting. This condition imposes a significant burden on affected individuals, their families, and society at large [1]. Moreover, chronic migraine often co-occurs with sleep disturbances, anxiety, and depression, compounding the overall health challenges faced by sufferers [2]. Existing international guidelines for migraine treatment include acute analgesic treatment and preventive treatment to reduce the frequency of headache attacks [3].

However, commonly prescribed preventive medications are known to have varying degrees of adverse effects. For instance, amitriptyline has potential adverse effects such as heart failure, contraindications with monoamine oxidase inhibitors and selective 5-hydroxytryptamine reuptake inhibitors, and an increased risk of glaucoma [4]. Flunarizine may lead to adverse effects like Parkinson’s disease and depression [5]. Valproate has potential adverse effects including liver disease, thrombocytopenia, and negative impacts on female fertility [2, 6, 7]. Novel CGRP or CGRP receptor blockade has demonstrated the effectiveness in migraine patients [8, 9]. Nonetheless, the use of monoclonal antibodies has limitations, including high cost and potential side effects like rash, itching, and pain [6]. As a result, there is an urgent need to discover a more affordable, safe, and efficacious medication for preventing migraine attacks.

Currently, the precise mechanism underlying migraine remains elusive. Recently, an increasing number of studies have shown that oxidative stress (OS) and neuroinflammation play a key role in the pathogenesis of migraine [10, 11]. Oxidative stress, which results from an imbalance between the production of reactive oxygen species (ROS) and the elimination of antioxidant defense mechanisms, is associated with various headache disorders, including migraine [12‐16], and endogenous ROS can cause oxidative damage to DNA, lipids, and proteins [17]. In contrast, reactive oxygen scavengers attenuated nociceptive hypersensitivity in rats with neuropathic pain, suggesting a possible role for antioxidants [18, 19]. Additionally, the role of inflammation in migraine is increasingly acknowledged. Xanthos, Sandkühler, and other researchers introduced the term "neurogenic neuroinflammation [20]." In the context of migraines, neurogenic neuroinflammation refers to the inflammatory response in both the central and peripheral components of the trigeminal nervous system to neuronal activity. Edvisson L and his colleagues suggest that the inflammatory process triggered by the continuous release of neurotransmitters may contribute to the chronicity of migraines [21]. Nucleus factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that is widely expressed in the central nervous system [22]. The study demonstrates that activating the Nrf2 signaling pathway can reduce oxidative stress and neuroinflammation [23, 24]. Other related research has shown that activation of the Nrf2/ARE pathway attenuates nociceptive sensitization in NTG-induced migraine mice [25, 26], suggesting that the Nrf2/ARE pathway may be a new therapeutic target for migraine.

DL-3-n-butylphthalide (NBP), a small-molecule compound independently developed from celery seed in China, was approved by the Chinese State Food and Drug Administration (SFDA) in 2005 for the treatment of ischemic stroke. It is relatively inexpensive with a very low incidence of adverse effects [27]. Numerous studies have documented that NBP exhibits the potential to mitigate oxidative stress and neuroinflammation through the activation of the Nrf2 pathway, with observed therapeutic benefits in animal models of Alzheimer's disease and depression [28, 29]. Nevertheless, it remains unexplored whether NBP can extend its therapeutic efficacy to the treatment of migraine.

In this study, we established a chronic migraine mouse model and administered NBP pre-treatment to simulate prophylactic treatment. Through this methodology, we hoped to evaluate the therapeutic effects of NBP through the behavioural performance of the mice, the expression levels of migraine-related markers, as well as oxidative stress and inflammatory factors. In addition, we also analyzed the Nrf2 pathway to further understand the mechanism of NBP treatment of migraine.

In the present study, we first developed an NTG-induced chronic migraine mouse model and confirmed the model’s reliability by measuring migraine-like behaviors and the abnormal expression of c-Fos and CGRP in the SP5C. Subsequently, we discovered that NBP pretreatment significantly ameliorated NTG-induced migraine-like behaviors and mitigated the increased expression of c-Fos and CGRP triggered by repeated NTG administration in mice. Meanwhile, NBP alleviated the enhanced oxidative stress and inflammation in the SP5C in NTG mice. Our experimental results further verified that NBP activates Nrf2 and its downstream targets. The administration of ML385 rendered NBP ineffective in alleviating migraine-like behaviors in NTG mice, indicating that the therapeutic efficacy of NBP hinges on the activation of the Nrf2/ARE pathway. Our research affirms that NBP activation of the Nrf2 pathway alleviates central sensitization in chronic migraine mice by mitigating oxidative stress and neuroinflammation.

NTG-treated mice are now a well-established and widely used experimental model of migraine, which can mimic many clinical features of migraine patients [33‐35]. Repeated injections of NTG resulted in a gradual decrease in mechanical nociceptive thresholds, an effect that was observed for up to one week following the cessation of NTG [33]. This hypersensitivity is indicative of cutaneous allodynia, which is present not only in the head but also in other areas of the body [36, 37]. In our present study, both periorbital and hindpaw cutaneous allodynia were observed, which suggest a severe state of central sensitization in NTG-treatment mice. Central sensitization is considered an important mechanism for the chronicity of migraine. Moreover, about 80% of migraineurs have concomitant symptoms of photophobia, and anxiety and depression [38]. Harris et al. have demonstrated photophobia and anxiety-like behaviour in NTG-induced animal models [39]. Here, we similarly assessed photophobia and anxiety-like behaviour to confirm the reliability of the model.

Photophobia is one of the most common symptoms of migraine, and the underlying mechanism is uncertain. The coexistence of photophobia and headache is associated with the interactions between visual and pain pathway at retina, midbrain, thalamus, hypothalamus and visual cortex. The communication between these pathways may depend on CGRP and pituitary cyclase-activating polypeptide transmission [40]. However, in our study, NBP improved the photophobic performance of CM mice, which may be achieved by lowering CGRP levels. Migraine is associated with a variety of psychiatric co-morbidities, including anxiety-depression, and the mechanisms involved are unclear [41]. Ruozhi Dang et al. reported that Edaravone attenuated oxidative stress through the Nrf2 pathway and alleviated anxiety-depressive behaviour in mice [42]. This may be consistent with the pathway by which NBP alleviated anxiety-depressive behaviour in CM mice in the present study.

The role of CGRP in migraine is well-established. It enhances nociceptive transmission and contributes to central sensitization by fostering and maintaining a hyper-responsive state [43]. The nuclear protein c-Fos is rapidly expressed in neurons in response to various types of noxious stimuli [44]. A study conducted by Shouyi Wu et al. evaluated NTG-induced migraine in mice by quantifying c-Fos and CGRP levels in SP5C and assessing central sensitization [45]. In our current study, we adopted a similar methodology to evaluate migraine, with an added focus on the expression levels of CGRP in peripheral blood. Our findings revealed that the changes in peripheral CGRP mirrored those observed in the central nervous system, corroborating the elevated CGRP levels found in the blood of migraine patients [9].

Within the known pathogenesis of migraine, oxidative stress and inflammation have been identified as universal triggers [11, 46, 47]. Riboflavin, known for its ability to reduce oxidative stress, has been found to prevent migraines [47]. The link between migraines and inflammation is still a hypothesis and has not been definitively confirmed. However, a growing body of research indicates a potential connection between inflammation and migraines. Numerous experiments, primarily conducted on rodent models, suggest that the nociceptive trigeminocervical complex, responsible for headaches, can be triggered by aseptic meningeal inflammatory processes [48]. Additionally, a study by Wei He et al. suggests that the activation of NLRP3 in microglia plays a role in the inflammatory responses associated with central sensitization of migraines in the NTG-induced CM mouse model [45]. Research by Rosaria Greco et al. demonstrates increased expression of pro-inflammatory cytokines in the TNC region of rats in the NTG-induced migraine model [49]. Furthermore, a PET-MRI study utilizing a highly sensitive inflammation tracer on 11 migraine patients has confirmed the connection between meningeal inflammation and migraines [50]. These collective findings suggest that although the precise relationship between migraines and inflammation remains unclear, inflammation may indeed contribute to the development of migraines. Consequently, strategies that target oxidative stress and inflammation present a viable approach for migraine management. Nrf2 is increasingly recognized as a key regulator of cellular defenses against oxidative stress. The activation of the Nrf2 signaling pathway serves to protect cells from oxidative stress and inflammation [24].

In China, NBP has been approved for the treatment of cerebral ischemia. However, numerous researchers have discovered that NBP also holds therapeutic potential in various neurological disorders. Mengqi Yang et al. showed that NBP may be able to treat major depressive disorder (MDD) [28]. Chun-Yan Wang et al. showed that NBP treatment could suppress TXNIP-NLRP3 interaction and inhibit NLRP3 inflammasome activation via upregulation of Nrf2 [51]. These findings suggest that NBP may have a role in activating Nrf2 expression and ameliorating the level of oxidative stress and inflammation, and in the present study, we found that NBP may ameliorate migraine through the same mechanism of action. Similarly, Wei Di et al. found that activation of the Nrf2 pathway ameliorates NTG-induced migraine in mice, which is consistent with our findings. They also found in a single administration of NTG-induced migraine mouse model, the expression of Nrf2 in the nucleus of cells in the SP5C was significantly increased compared to the Control + group [25], which may be due to the fact that NTG injections may enhance the antioxidant capacity of cells to cope with nitric oxide-induced oxidative stress by inducing the expression of Nrf2, causing a compensatory mechanism. However, our results showed that repeated NTG injections led to a decrease in nuclear Nrf2 expression, which may be due to the fact that multiple NTG injections mimic the chronicity of migraine, in which the body's antioxidant capacity is insufficient to combat peroxidation, resulting in a state of loss of compensation. In addition, some studies of other chronic neurological disorders, such as Amyotrophic Lateral Sclerosis (ALS) and MDD, have also shown a decrease in Nrf2 expression in the animal model group [28, 52], which is consistent with our findings.

Furthermore, several studies have shown that the Nrf2/ARE pathway not only regulates oxidative stress but also attenuates inflammation [24, 53, 54], as the HO-1 axis regulated by Nrf2 is a potent anti-inflammatory target [53]. During our experiments, we found that NBP attenuated the high expression of pro-inflammatory factors and improved some indicators of oxidative stress. These results suggest that NBP may alleviate migraine in mice by reducing oxidative stress and neuroinflammation, but the specific anti-inflammatory mechanism of Nrf2 needs further investigation. ML385, a cancer-fighting molecule commonly used in tumor research, specifically inhibits the binding of Nrf2 to DNA, thereby suppressing the expression of Nrf2 [55], and previous studies have shown that intraperitoneal administration of ML385 at 30 mg/kg can significantly inhibit Nrf2 activation in mice [31, 32]. After administration of ML385 to block Nrf2 expression, we found that NBP failed to improve migraine-like behavioural in mice, which may indicate that NBP treatment is dependent on the Nrf2/ARE pathway. Nonetheless, it is important to address one of the limitations of our study, which is that we did not further substantiate this idea by deeper molecular studies. This is something we intend to focus on in our future and complementary studies. Meanwhile, the application of ML385 had no effect on the behaviour of normal mice, which may be due to the fact that the level of oxidative stress in normal mice is at a plateau, and inhibition of Nrf2 does not affect the normal level of oxidative stress.

In summary, our study highlights the significant preventive therapeutic potential of NBP in a mouse model of chronic migraine. This suggests that NBP could serve as an effective agent for preventing migraine attacks, a prospect that is particularly promising given the limitations of existing prophylactic medications for migraine. Moreover, the anti-inflammatory and antioxidant properties of NBP, as demonstrated in our study, suggest that NBP could also be effective in the acute treatment of migraine and other pain disorders. This possibility is intriguing and warrants further investigation in our future research. In conclusion, our study not only provides a effective strategy for managing migraines, but also broadens the potential applications of NBP in other medical conditions.

In summary, our study demonstrated that NBP effectively attenuated NTG-induced migraine in mice. It exerted its therapeutic effects by mitigating nociceptive hypersensitivity and central sensitization through the activation of the Nrf2 signaling pathway, thereby reducing oxidative stress and neuroinflammation. Consequently, NBP holds promising potential as a prophylactic treatment for migraine.